Calcium channels are membrane-spanning, multisubunit proteins that allow controlled entry of Ca.sup.+2 ions into cells from the extracellular fluid. All cells throughout the animal kingdom, and at least some bacterial, fungal and plant cells, possess one or more types of calcium channel.
The most common type of calcium channel is voltage-dependent. In a voltage-dependent channel, the "opening" which allows there to begin an influx of Ca.sup.+2 ions into the cells requires a depolarization to a certain level of the potential difference between the inside of the cell bearing the channel and the extracellular medium bathing the cell. The rate of influx of Ca.sup.+2 into the cell depends on this potential difference. All "excitable" cells in animals, such as neurons of the central nervous systems, peripheral nerve cells, and muscle cells, including those of skeletal muscles, cardiac muscles, and venous and arterial smooth muscles, have voltage-dependent calcium channels.
Calcium channels are physiologically important because the channels have a central role in regulating intracellular Ca.sup.+2 levels, which levels are important for cell viability and function. Thus, intracellular Ca.sup.+2 concentrations are implicated in a number of vital processes in animals, such as neurotransmitter release, muscle contraction, pacemaker activity, and secretion of hormones and other substances.
A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-dependent calcium channels. Many of these compounds bind to calcium channels and block, or reduce the rate of, influx of Ca.sup.+2 into cells in response to depolarization of the inside and outside of the cells.
An understanding of the pharmacology of compounds that interact with calcium channels, and the ability to rationally design compounds that will interact with calcium channels to have desired therapeutic effects, have been hampered by a lack of understanding of the structure of channel subunits and the genes that code for them. Thus, it has not been possible to obtain the large amounts of highly purified channel subunits that are required to understand, at the molecular level, the nature of the subunits and their interactions with one another, with the cell membranes across which the channels allow Ca.sup.+2 ions to pass, with Ca.sup.+2 and other ions, and with low molecular weight compounds that affect channel function. For example, with the availability of large amounts of purified calcium channel subunits, functional channels could be prepared and used to screen the effects of compounds on channel function, thereby providing a basis for the design of therapeutic agents which affect the calcium channel, or various combinations of channel subunits could be crystallized and have their structures determined to high resolution employing X-ray or neutron diffraction techniques, providing yet another basis for rational design of therapeutic agents that affect channel function.
Certain diseases, such as Lambert-Eaton Syndrome, involve autoimmune interactions with calcium channels. The ready availability of each of the calcium channel subunits would make possible immunoassays for the diagnosis of such diseases and an understanding of them at the molecular level that could lead to effective methods of treatment.
The lack of information on genes that code for calcium channel subunits has prevented the understanding of the molecular properties of the mature calcium channel subunits and their precursor proteins (i.e., the mature subunits with signal peptides appended to the amino-terminus) and the regulation of expression of calcium channel subunits. An understanding of these properties, and of how expression of calcium channel subunit genes is regulated, may provide the basis for designing therapeutic agents which have beneficial effects through affecting calcium channel function or concentration. Furthermore, the availability of sequences of genes coding for calcium channel subunits would make possible the diagnosis of defects in genes coding for such subunits, which might underlie a number of diseases.
The availability of a DNA with the sequence of a segment of at least about 14 and more preferably at least about 30 nucleotides of a cDNA encoding a subunit of a calcium channel from the cells of a tissue of an animal would make possible the isolation and cloning of cDNAs, and possibly genomic DNAs, coding for the corresponding subunit of different calcium channels from the same or different tissues and animals of the same or different species. The availability of the sequences of numerous full-length cDNAs coding for corresponding subunits of calcium channels from a variety of tissues and animal species would contribute to elucidating structure-function relationships in the subunits. This knowledge, in turn, would be useful in the design of therapeutic agents whose activities are exerted through binding to calcium channels.
In skeletal muscle, where voltage-dependent calcium channels have been best characterized, voltage-dependent calcium channels are thought to consist of two large subunits, of between about 130 and about 200 kilodaltons ("kD") in molecular weight, and a number (generally thought to be two or three) of different smaller subunits, of less than about 60 kD in molecular weight. At least one of the larger subunits and possibly some of the smaller are glycosylated. Some of the subunits are capable of being phosphorylated. There has been confusion in the art concerning the naming of the various subunits of voltage-dependent calcium channels.
The two large subunits of voltage-dependent calcium channels are designated herein the ".alpha..sub.1 -subunit" and the ".alpha..sub.2 -subunit".
The .alpha..sub.1 -subunit is not detectably changed in molecular weight when treated with dithiothreitol ("DTT") or with enzymes which catalyze removal of N-linked sugar groups from glycosylated proteins. The .alpha..sub.1 -subunit has a molecular weight of about 150 to about 170 kD when analyzed by sodium dodecylsulfate ("SDS")-polyacrylamide gel electrophoresis ("PAGE") after isolation from mammalian muscle tissue and has specific binding sites for various 1,4-dihydropyridines ("DHPs") and phenylalkylamines.
The .alpha..sub.2 -subunit is somewhat less well characterized than the .alpha..sub.1 -subunit, although recent work by Ellis et al. (see PCT Application No. WO 89/09834 and Science, 241, 1661-1664 (1988)), has provided a great deal of additional information concerning this subunit. The molecular weight of the .alpha..sub.2 -subunit is at least about 130-150 kD, as determined by SDS-PAGE analysis in the presence of DTT after isolation from mammalian muscle tissue. However, in SDS-PAGE under non-reducing conditions (in the presence of N-ethylmaleimide), the .alpha..sub.2 -subunit migrates with a band of about 160-190 kD. It is presently believed that the smaller fragment (of about 30 kD), which appears to be released upon reduction of the .alpha..sub.2 -subunit, is the carboxy terminus of the primary translation product of the .alpha..sub.2 -subunit mRNA. Regardless, however, of whether the two fragments are different subunits of the calcium channel or whether both fragments are products of the same gene (and, consequently, the .alpha..sub.2 -subunit is about 160-190 kD and is split into (at least) two fragments upon reduction), there is evidence that the .alpha..sub.2 -subunit, whatever its size, and the corresponding fragment produced under reducing conditions, whether part of the .alpha..sub.2 -subunit or not, are glycosylated with at least N-linked sugars. In addition there is evidence that the .alpha.2-subunit and the corresponding fragment produced under reducing conditions do not have specified binding sites for 1,4-dihydropyridines and phenylalkylamines, which species are known to bind to the .alpha..sub.1 -subunit.
The .beta.-subunit of the calcium channel has only recently been characterized as having an apparent molecular mass of 52-65 kD (as determined by SDS-PAGE analysis). It is comprised of consensus phosphorylation sites and has been shown by biochemical methods to be phosphorylated. This subunit is insensitive to reducing conditions.
The .gamma.-subunit of the calcium channel has not been observed in all purified preparations, depending on the source of material analyzed, the investigating laboratory, and so on. Because of its irregular appearance in the hands of some investigators, this particular subunit also remains relatively poorly characterized. The native material appears to be a glycoprotein with an apparent molecular mass of 30-33 kD, as determined by SDS-PAGE analysis. The native protein is believed to be glycosylated since its apparent molecular mass decreases after digestion with neuraminidase followed by endoglycosidase F.
Reference herein to the precursor of an .alpha..sub.1 -subunit means the protein with the amino acid sequence corresponding to the sequence of the full-length mRNA which, upon translation, results, ultimately, in .alpha..sub.1 -subunit resident as part of a calcium channel in a cell membrane. The precursor protein is converted by various, not presently well understood, processing steps to produce the .alpha..sub.1 -subunit.
Similarly, reference herein to the precursor of an .alpha..sub.2 -subunit means the protein with the amino acid sequence corresponding to the sequence of the full-length mRNA which, upon translation, results, ultimately, in .alpha..sub.2 -subunit resident as part of a calcium channel in a cell membrane. The precursor protein is converted by various processing steps into the .alpha..sub.2 -subunit. As with the .alpha..sub.1 -subunit, the details of the processing between the precursor and the mature .alpha..sub.2 -subunit are not clear, but the processing presumably involves at least removal of a leader sequence (i.e., a signal peptide), glycosylation, and cleavage to yield what is now thought to be .delta.-subunit of the calcium channel.
Similarly, reference herein to the precursor of a .beta.-subunit means the protein with the amino acid sequence corresponding to the sequence of the full-length mRNA which, upon translation, results ultimately, in .beta.-subunit resident as part of a calcium channel in a cell membrane. The precursor protein is converted by various, not presently well understood, processing steps into the .beta.-subunit.
Similarly, reference herein to the precursor of a .gamma.-subunit means the protein with the amino acid sequence corresponding to the sequence of the full-length mRNA which, upon translation, results ultimately, in .gamma.-subunit resident as part of a calcium channel in a cell membrane. The precursor protein is converted by various, not presently well understood, processing steps into the .gamma.-subunit.
The cDNA and corresponding amino acid sequence of the .alpha..sub.1 -subunit precursor of a rabbit back skeletal muscle calcium channel has been reported. See Tanabe et al., Nature 328, 313-318 (1987).
The cDNA and corresponding amino acid sequences of the .alpha..sub.2 -subunit precursor of a rabbit back skeletal muscle calcium channel and a human neuronal calcium channel have also been reported. See Ellis, et al., PCT Application No. WO 89/09834 (1989) and Science 241, 1661-1664 (1988).
The cDNA and corresponding amino acid sequence of the .beta.-subunit precursor of a rabbit back skeletal muscle calcium channel has also been reported. See Ruth et al., Science 245, 1115-1118 (1989).
Up to now, however, the cDNA and corresponding amino acid sequence of the .gamma.-subunit precursor of a calcium channel have not been reported in the literature.
Calcium channel activity, measured electrophysiologically by voltage-clamp techniques, has been induced in Xenopus laevis oocytes when total mRNA isolated from mammalian brain or cardiac muscle is injected into the oocytes. Also, it has been reported that calcium channel-containing preparations, when reconstituted into lipid bilayers, confer voltage-dependent calcium channel activity on the bilayers.
However, there is no evidence that any one of the calcium channel subunits alone provides a natively functional calcium channel in oocytes, lipid bilayers or any other situation. It has been recently reported by Hofmann, et al., Trends in Pharmacology. Sci. 8, 393-398 (1987) that mRNA prepared using the cDNA of .alpha..sub.1 -subunit obtained by Tanabe, et al. was unable to induce calcium channel activity in Xenopus laevis oocytes.